Bioadhesive for occluding vessels

ABSTRACT

Bioadhesives and crosslinked gels therefrom are disclosed. The bioadhesives can be applied to a vessel for occluding the vessel. The present disclosure also describes kits that comprise the various components for preparing and applying the bioadhesives. Bioadhesives of the present disclosure include: (i) a biopolymer having one or more first chemically reactive amine groups; (ii) a biocompatible crosslinker having at least two second chemically reactive groups that can chemically react with the one or more first chemically reactive amine groups of the biopolymer; and (iii) a biocompatible rheological modifier.

TECHNICAL FIELD

The present disclosure relates to a bioadhesive and crosslinked gelstherefrom and to making and using the bioadhesive for occluding vessels.

BACKGROUND

Bioadhesives can be used to seal blood vessels. However, fluid at thesite of application, such as blood, may prevent the bioadhesive fromworking properly. For example, in a vessel, blood may dilute thebioadhesive or wash it away entirely before the bioadhesive has anopportunity to adhere and/or crosslink.

One place it may be useful to use bioadhesives is within vessels, suchas blood vessels. For example, spider veins (telangiectasias) are causedwhen small, superficial blood vessels dilate and rise above the skinsurface, appearing most commonly on the face and legs. They can be red,purple or bluish in color, and can appear in noticeable small patches orcan cover large areas of skin.

Telangiectasia develops in the legs often due to the presence of venoushypertension within underlying varicose veins or underlying venousreflux disease. Flow abnormalities within the medium sized veins of theleg (reticular veins) can also lead to the development oftelangiectasia.

Current treatments for such modalities include the use of commercialsclerosants, e.g., sodium tetradecyl sulfate (STS) and polidocanol,which are injected into the vein and work by damaging the cell lining ofblood vessels, causing them to close and eventually be replaced by othertypes of tissue. Such current treatment can require multiple sessions,and in some case can result in long term staining.

Biomaterial based treatments are also being proposed (but not approvedfor use) for spider vein treatment. The limitations of biomaterials areuncontrolled degradation of the biomaterial, weak adhesion, andinsufficient or slow absorption of the vein into surrounding tissue.Some of these limitations may be caused by blood flowing through thevessel, thereby diluting or washing the biomaterial away before thebiomaterial has an opportunity to properly cure. Further, biomaterialbased treatments can also suffer from poor cosmetic effects (such asinflammation and necrosis and staining). Accordingly, a need exists forimproved formulations that can occlude vessels in a mammal.

SUMMARY OF THE DISCLOSURE

An advantage of the present disclosure is a bioadhesive that can bereadily used to occlude vessels, e.g., blood vessels of a mammal. Thebioadhesive of the present disclosure may be implemented to realize oneor more of the following advantages. The bioadhesive of the presentdisclosure has viscosity sufficient to displace fluids in a vessel, suchas blood, while the bioadhesive crosslinks, but is still capable ofbeing injected through a thin gauge needle. The bioadhesive crosslinksin place while preventing fluids, such as blood, from pushing back intothe treatment area and washing the bioadhesive away before thebioadhesive has an opportunity to crosslink. Further, once crosslinked,the gel formed from the bioadhesive can have a color that cannot be seenthrough the skin, and can have a tactile feel similar to the vessel intowhich the bioadhesive was injected, thereby providing an immediatecosmetic and physiological effect.

These and other advantages are satisfied, at least in part, by abioadhesive comprising: (i) a biopolymer having one or more firstchemically reactive amine groups; (ii) a biocompatible crosslinkerhaving at least two second chemically reactive groups that canchemically react with the one or more first chemically reactive aminegroups; and (iii) a biocompatible rheological modifier.

Embodiments include one or more of the following features individuallyor combined. For example, the biopolymer can be a protein or apolysaccharide having one or more primary amines as the first chemicallyreactive groups. In other embodiments, the biopolymer is albumin. Insome embodiments, the protein is collagen and the polysaccharide is achitosan having one or more first chemically reactive amine groups. Inother embodiments, the biopolymer is a synthetic polymer selected fromthe group consisting of amine functionalized polyethylene glycols,polyallylamine, and branched polyethylenimine. In some embodiments, thebiocompatible crosslinker is a multi-arm polyethylene glycol (PEG)having at least two or more N-hydroxysuccinimide (NHS) ester groups asthe second chemically reactive groups. In still further embodiments, thebiocompatible rheological modifier can be a shear thinning fluid with anon-sheared viscosity between 0.5 Pa·s and 200 Pa·s. In someembodiments, the biocompatible rheological modifier is hyaluronic acidor a salt thereof, such as sodium hyaluronate. In other embodiments, thebiopolymer is albumin having one or more primary amines as the firstchemically reactive amine groups, the biocompatible crosslinker is amulti-arm PEG having at least two or more N-hydroxysuccinimide estergroups as the second chemically reactive groups and the biocompatiblerheological modifier is sodium hyaluronate. In various embodiments, theconcentration of the biopolymer in the bioadhesive is between 7.5 wt %to 5 wt %, and the bioadhesive has a pH of less than 7.4.

In accordance with the present disclosure, a bioadhesive can comprise:(i) a biopolymer having one or more first chemically reactive aminegroups; (ii) a biocompatible crosslinker having at least two secondchemically reactive groups that can chemically react with the one ormore first chemically reactive amine groups; and (iii) a biocompatiblerheological modifier, wherein the concentration of the biopolymer in thebioadhesive is less than 15% by weight.

Embodiments include one or more of the following features individuallyor combined. For example, the biopolymer can be a protein or apolysaccharide having one or more primary amines as the first chemicallyreactive amine groups. The biocompatible crosslinker can be a multi-armPEG having at least two or more N-hydroxysuccinimide (NHS) ester groupsas the second chemically reactive groups, for example. In otherembodiments, the biopolymer is an albumin having one or more primaryamines as the first chemically reactive amine groups, the biocompatiblecrosslinker is a multi-arm PEG having at least two or moreN-hydroxysuccinimide ester groups as the second chemically reactivegroups and the biocompatible rheological modifier is sodium hyaluronate.

In accordance with the present disclosure, a bioadhesive can comprise:(i) a biopolymer having one or more first chemically reactive aminegroups; (ii) a biocompatible crosslinker having at least two secondchemically reactive groups that can chemically react with the one ormore first chemically reactive amine groups; and (iii) a biocompatiblerheological modifier, wherein the bioadhesive has a gelation time ofmore than 5 minutes.

Embodiments include one or more of the following features individuallyor combined. For example, the biopolymer can be a protein or apolysaccharide having one or more primary amines as the first chemicallyreactive amine groups. In various embodiments, the biocompatiblecrosslinker is a multi-arm PEG having at least two or moreN-hydroxysuccinimide (NHS) ester groups as the second chemicallyreactive groups. In still further embodiments, the biopolymer is analbumin having one or more primary amines as the first chemicallyreactive amine groups, the biocompatible crosslinker is a multi-arm PEGhaving at least two or more N-hydroxysuccinimide ester groups as thesecond chemically reactive groups and the biocompatible rheologicalmodifier is sodium hyaluronate.

Another aspect of the present disclosure includes a method of preparinga bioadhesive and the gel therefrom. The method comprises: combining (i)a biopolymer having one or more first chemically reactive amine groups;(ii) a biocompatible crosslinker having at least two second chemicallyreactive groups that can chemically react with the one or more firstchemically reactive amine groups; and (iii) a biocompatible rheologicalmodifier, to form a bioadhesive. Advantageously, the biopolymercrosslinks with the biocompatible crosslinker to form a bioadhesive gel.

Embodiments include one or more of the following features individuallyor combined. For example, the method can include transferring thebioadhesive to a syringe suitable for injecting the bioadhesive into avessel of a mammal. In some embodiments, the bioadhesive has a gelationtime of more than 5 minutes. In some embodiments, the biopolymer isalbumin. In other embodiments, the biopolymer is chitosan or thebiopolymer is a synthetic polymer selected from the group consisting ofamine functionalized polyethylene glycols, and branchedpolyethylenimine. In still further embodiments, the biocompatiblecrosslinker is a multi-arm PEG having at least two or moreN-hydroxysuccinimide (NHS) ester groups as the second chemicallyreactive groups. In some embodiments, the biocompatible rheologicalmodifier can be a shear thinning fluid with a non-sheared viscositybetween 0.5 Pa·s and 200 Pa·s. In various embodiments, the biocompatiblerheological modifier is hyaluronic acid or a salt thereof, such assodium hyaluronate. In still further embodiments, the biopolymer is analbumin having one or more primary amines as the first chemicallyreactive amine groups, the biocompatible crosslinker is a multi-arm PEGhaving at least two or more N-hydroxysuccinimide ester groups as thesecond chemically reactive groups, and the biocompatible rheologicalmodifier is sodium hyaluronate. Additional embodiments include mixingthe biopolymer and the biocompatible rheological modifier in a firstcontainer; mixing the biocompatible crosslinker in a buffer solution ina second container; and mixing the contents of the first container andthe second container together to form the bioadhesive, and wherein thebuffer solution has a pH between 4.5 and 7.4.

In accordance with the present disclosure, a method of preparing abioadhesive and gel therefrom includes combining (i) a formulationincluding a biopolymer having one or more first chemically reactiveamine groups and a biocompatible rheological modifier with (ii) aformulation including a biocompatible crosslinker having at least twosecond chemically reactive groups that can chemically react with the oneor more first chemically reactive amine groups, to form a bioadhesivewherein the biopolymer crosslinks with the biocompatible crosslinker toform a bioadhesive gel.

Embodiments include one or more of the following features individuallyor combined. For example, the biopolymer can be albumin. In someembodiments, the biocompatible crosslinker is a multi-arm PEG having atleast two or more N-hydroxysuccinimide (NHS) ester groups as the secondchemically reactive groups. In some embodiments, the biocompatiblerheological modifier is hyaluronic acid or a salt thereof.

In accordance with the present disclosure, a method of preparing abioadhesive and gel therefrom includes combining (i) a formulationincluding a biopolymer having one or more first chemically reactiveamine groups and a biocompatible rheological modifier with (ii) aformulation including a buffer and a biocompatible crosslinker having atleast two second chemically reactive groups that can chemically reactwith the one or more first chemically reactive amine groups, to form abioadhesive wherein the biopolymer crosslinks with the biocompatiblecrosslinker to form a bioadhesive gel.

Embodiments include one or more of the following features individuallyor combined. For example, the biopolymer can be albumin. In someembodiments, the biocompatible crosslinker is a multi-arm PEG having atleast two or more N-hydroxysuccinimide (NHS) ester groups as the secondchemically reactive groups. In some embodiments, the biocompatiblerheological modifier is hyaluronic acid or a salt thereof. In stillfurther embodiments, the buffer has a pH between 4.5 and 7.4. Additionalembodiments further include the biocompatible rheological modifier is ashear thinning fluid with a non-sheared viscosity between 0.5 Pa·s and200 Pa·s.

Another aspect of the present disclosure includes a method of occludinga vessel of a mammal. The method comprises injecting a bioadhesive intoa vessel of a mammal which crosslinks in the vessel to occlude thevessel, wherein the bioadhesive comprises: (i) a biopolymer having oneor more first chemically reactive amine groups; (ii) a biocompatiblecrosslinker having at least two second chemically reactive groups thatcan chemically react with the one or more first chemically reactiveamine groups of the biopolymer; and (iii) a biocompatible rheologicalmodifier.

Embodiments include one or more of the following features individuallyor combined. For example, the biocompatible rheological modifier canhave a viscosity sufficient to displace fluid within the vessel whilethe biopolymer and biocompatible crosslinker crosslink. In someembodiments, the biocompatible rheological modifier is a shear thinningfluid with a non-sheared viscosity between 0.5 Pa·s and 200 Pa·s.Additional embodiments include injecting the bioadhesive into the vesselby a syringe. In various embodiments, the bioadhesive can be prepared bycombining the biopolymer, the biocompatible crosslinker and thebiocompatible rheological modifier prior to injecting the bioadhesiveinto the vessel. The bioadhesive can further comprise a buffer, forexample. In some embodiments, the bioadhesive has a gelation time ofbetween 0.5 minute and 40 minutes. Additional embodiments includeinjecting the bioadhesive into a blood vessel of a mammal having adiameter of between 0.3 mm to 6 mm, or injecting the bioadhesive into ablood vessel of a mammal having a diameter of greater than 6 mm. Instill further embodiments, the rheological modifier displaces blood andprevents blood flow back into the blood vessel while the biopolymercrosslinks with the biocompatible crosslinker. Advantageously, therheological modifier is absorbed into tissue surrounding the bloodvessel after the bioadhesive has crosslinked. In various embodiments,the vessel is absorbed into surrounding tissue in a 4 to 8 week periodafter injection of the bioadhesive into the vessel. In still furtherembodiments, the biopolymer is an albumin or a chitosan. In someembodiments, the biocompatible crosslinker is a multi-arm PEG having atleast two or more N-hydroxysuccinimide (NHS) ester groups as the secondchemically reactive groups. In some embodiments, the biocompatiblerheological modifier is hyaluronic acid or a salt thereof such as sodiumhyaluronate. In still further embodiments, the biopolymer is albuminhaving one or more primary amines as the first chemically reactive aminegroups, the biocompatible crosslinker is a multi-arm PEG having at leasttwo or more N-hydroxysuccinimide ester groups as the second chemicallyreactive groups, and the biocompatible rheological modifier is sodiumhyaluronate.

In accordance with the present disclosure, a method of occluding a bloodvessel of a mammal includes: preparing a bioadhesive by combining (i) aformulation including a biopolymer having one or more first chemicallyreactive amine groups and a biocompatible rheological modifier with (ii)a formulation including a biocompatible crosslinker having at least twosecond chemically reactive groups that can chemically react with the oneor more first chemically reactive amine groups; and injecting thebioadhesive into a blood vessel which crosslinks in the blood vessel toocclude the blood vessel.

Embodiments include one or more of the following features individuallyor combined. For example, the method can include injecting thebioadhesive into a blood vessel having a diameter between 0.3 mm to 6mm. The bioadhesive can have a gelation time of between 5 minutes and 40minutes, for example. In some embodiments, the biopolymer is albumin. Insome embodiments, the biocompatible rheological modifier is sodiumhyaluronate, and the biocompatible crosslinker is a multi-arm PEG havingat least two or more N-hydroxysuccinimide ester groups as the secondchemically reactive groups. Additional embodiments include preparing theformulation including the biocompatible crosslinker by combining amulti-arm PEG as the biocompatible crosslinker with a buffer. In someembodiments, the buffer has a pH of between 4.5 and 7.4. In someembodiments, the method includes injecting the biocompatible formulationinto a blood vessel having a diameter greater than 6 mm.

In accordance with the present disclosure, a method of occluding a bloodvessel of a mammal includes: preparing a bioadhesive by combining (i) aformulation including a biopolymer having one or more first chemicallyreactive amine groups and a biocompatible rheological modifier with (ii)a formulation including a buffer and a biocompatible crosslinker havingat least two second chemically reactive groups that can chemically reactwith the one or more first chemically reactive amine groups; andinjecting the bioadhesive into a blood vessel which crosslinks in theblood vessel to occlude the blood vessel.

Embodiments include one or more of the following features individuallyor combined. For example, the biocompatible rheological modifier can bea shear thinning fluid with a non-sheared viscosity between 0.5 Pa·s and200 Pa·s. The method can include injecting the biocompatible formulationinto a blood vessel having a diameter of between 0.3 mm to 6 mm, forexample. In some embodiments, the bioadhesive has a gelation time ofbetween 5 minutes and 40 minutes. In some embodiments, the buffer has apH between 4.5 and 7.4. In still further embodiments, the rheologicalmodifier is absorbed into tissue surrounding the blood vessel after thebioadhesive has crosslinked. Additional embodiments include wherein theinjected bioadhesive displaces blood in the blood vessel and preventsblood flow back into the blood vessel while the biopolymer andbiocompatible crosslinker crosslink in the blood vessel.

Another aspect of the present disclosure includes a kit comprising: (i)a first container including a biopolymer having one or more firstchemically reactive amine groups; (ii) a second container including abiocompatible crosslinker having at least two second chemically reactivegroups that can chemically react with the one or more first chemicallyreactive amine groups of the biopolymer; and (iii) a biocompatiblerheological modifier included either in the first container or thesecond container or in a third container.

Embodiments include one or more of the following features individuallyor combined. For example, the biopolymer can be a protein or apolysaccharide having one or more primary amines as the first chemicallyreactive amine groups. In other embodiments, the biopolymer is albumin.In still further embodiments, the biopolymer is collagen. The biopolymercan further be a chitosan having one or more first chemically reactiveamine groups in some embodiments or a synthetic polymer selected fromthe group consisting of amine functionalized polyethylene glycols,polyallylamine, and branched polyethylenimine in other embodiments. Insome embodiments, the biocompatible crosslinker is a multi-arm PEGhaving at least two or more N-hydroxysuccinimide (NHS) ester groups asthe second chemically reactive groups. In various embodiments, thebiocompatible rheological modifier is a shear thinning fluid with anon-sheared viscosity between 0.5 Pa·s and 200 Pa·s. In someembodiments, the biocompatible rheological modifier is hyaluronic acidor a salt thereof such as sodium hyaluronate. In further embodiments,the biopolymer is albumin having one or more primary amines as the firstchemically reactive amine groups, the biocompatible crosslinker is amulti-arm PEG having at least two or more N-hydroxysuccinimide estergroups as the second chemically reactive groups, and the biocompatiblerheological modifier is sodium hyaluronate. In some embodiments, thebiopolymer and the biocompatible rheological modifier are included as aformulation in the first container. In some embodiments, thebiocompatible crosslinker is contained in the second container as aformulation having a pH between 4.5 and 7.4. Additional embodimentsinclude, wherein when the contents of the first container, the secondcontainer and the biocompatible rheological modifier of the kit arecombined to form a formulation, the concentration of the biopolymer inthe formulation is 15 wt % or less.

In accordance with the present disclosure, a kit can include: (i) afirst container including a biopolymer having one or more firstchemically reactive amine groups; (ii) a second container including abiocompatible crosslinker having at least two second chemically reactivegroups that can chemically react with the one or more first chemicallyreactive amine groups of the biopolymer; (iii) a third containerincluding a buffer solution; and (iv) a biocompatible rheologicalmodifier included in any one of the first, second or third containers orin a fourth container.

Embodiments include one or more of the following features individuallyor combined. For example, the biocompatible crosslinker can be amulti-arm polyethylene glycol having at least two or moreN-hydroxysuccinimide (NHS) ester groups as the second chemicallyreactive groups; and the buffer solution includes citric acid and sodiumphosphate dibasic in water for injection (WFI) grade water and has a pHof between 4.5 and 7.4. Other embodiments include wherein the biopolymeris an albumin, and the first container includes sodium hyaluronate asthe biocompatible rheological modifier formulated in a buffer solutionat a concentration of less than 2%. Additional embodiments includewherein when the contents of the first container, the second container,the third container and the biocompatible rheological modifier of thekit are combined to form a formulation, the concentration of thebiopolymer in the formulation is between 5% and 7.5% by weight.

In accordance with the present disclosure, a kit can include: (i) afirst container including albumin having one or more first chemicallyreactive amine groups; (ii) a second container including a multi-armpolyethylene glycol having at least two second chemically reactivegroups that can chemically react with the one or more first chemicallyreactive amine groups of the albumin; (iii) a hyaluronate saltrheological modifier included either in the first container or thesecond container or in a third container.

Embodiments include one or more of the following features individuallyor combined. For example, the kit can include a first syringe as thefirst container. The first syringe can include the albumin andhyaluronate salt rheological modifier as a formulation, and the secondcontainer can be a second syringe including the multi-arm polyethyleneglycol as a formulation having a pH of between 4.5 and 7.4. Additionalembodiments include wherein the multi-arm polyethylene glycolformulation includes citric acid, dibasic phosphate solution and WFIgrade water.

Additional advantages of the present invention will become readilyapparent to those skilled in this art from the following detaileddescription, wherein only the preferred embodiment of the invention isshown and described, simply by way of illustration of the best modecontemplated of carrying out the invention. As will be realized, theinvention is capable of other and different embodiments, and its severaldetails are capable of modifications in various respects, all withoutdeparting from the invention. Accordingly, the drawings and descriptionare to be regarded as illustrative in nature, and not as restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

Reference is made to the attached drawings, wherein elements having thesame reference numeral designations represent similar elementsthroughout and wherein:

FIG. 1 is schematic illustration of a bioadhesive gel of the presentdisclosure within a blood vessel.

FIG. 2 is a chart plotting the force needed to eject a bioadhesive ofthe present disclosure at a rate of about 1.5 ml/min from a one (1) mlsyringe through a 30 gauge needle versus time and showing the gelationtime for the particular formulation.

FIG. 3 is a chart plotting force versus time and showing the effect ofpH on gelation time for bioadhesives of the present disclosure.

FIG. 4 is a chart showing the effects of pH and biopolymer concentrationon gelation time of bioadhesives of the present disclosure.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure relates to bioadhesives and crosslinked gelstherefrom. The bioadhesives can be inserted into a vessel of a mammal,e.g., a human, for occluding the vessel. The present disclosure alsodescribes kits that comprise the various components for preparing andapplying the bioadhesives. The bioadhesive of the present disclosureinclude: (i) a biopolymer having one or more first chemically reactiveamine groups; (ii) a biocompatible crosslinker having at least twosecond chemically reactive groups that can chemically react with the oneor more first chemically reactive amine groups of the biopolymer; and(iii) a biocompatible rheological modifier.

The biopolymer can be in a formulation, e.g., an aqueous formulation,alone or with other additives such as preservatives and/or pH modifiers.Also, the biocompatible crosslinker can be in a formulation, e.g., anaqueous formulation, either alone or with other additives such aspreservatives and/or pH modifiers in the formulation. The biocompatiblerheological modifier can also be in a separate formulation, e.g., anaqueous formulation, alone or with other additives such as preservativesand/or pH modifiers. Alternatively, the biocompatible rheologicalmodifier can be included in the formulation with either or both of thebiopolymer formulation and/or the crosslinker formulation.

The rheological modifier is provided in the formulation to increase theviscosity of the formulation. The increased viscosity of the formulationis advantageous when inserting the bioadhesive into a blood vessel sincethe biocompatible rheological modifier facilitates displacement of bloodin the vessel and prevents blood flow back into the vessel while thebiopolymer and crosslinker form a crosslinked network and adheres to thevessel. Similarly, in other vessels, such as the vas deferens orfallopian tube in a sterilization procedure, the rheological modifierfacilitates displacing any fluid that may be present to dilute or washaway the bioadhesive while the bioadhesive crosslinks. While increasingviscosity, the rheological modifier also has shear thinning propertiesto enable injection of the bioadhesive through a thin gauge needle orcatheter.

In one aspect of the present disclosure, the bioadhesive can be used inthe treatment of superficial or spider blood vessels. The formulationscan also be broadly used to treat larger blood vessels (perforators,varicose veins, arteries), vascular malformations (aneurysms, AVMs,fistula sealant, etc.), and for wound closure applications.

Another aspect of the bioadhesives of the present disclosure is that theformulation can be prepared to gel over a period that is appropriate forspecific procedures, such as procedures to occlude a vessel. As usedherein, gelation time is defined as the time when a formulation beingsto transform from a sol to a gel. With respect to a bioadhesive, thegelation time is the time period from when all the reactive componentsof the bioadhesive are combined to form a sol which transforms to a gel.The gelation time can be quantified as follows. A bioadhesive is mixedin a large syringe and transferred to several 1 mL syringes, and thenextruded through a 30G needle at a fixed flow rate (1.5 mL/min). Theforce needed to extrude the bioadhesive is quantified for one of thepremixed syringes at various time points and plotted. The data is fittedto a sigmoidal curve and gelation occurs as the fitted lines transitionto concave up. Gelation is estimated by the intersection of the tangentsto the points immediately before and after the transition. For example,FIG. 3 illustrates the gelation time for three bioadhesives. Theformulations were mixed in ten 1 mL syringes for each bioadhesive,extruded through a 30G needle at a fixed flow rate of 1.5 mL/min, andthe force was measured over time (i.e., one syringe was measured at eachspecific time point, and each data point represents one syringe). Thedata was fitted to sigmoidal curves for each formulation. As can be seenin FIG. 3, the bioadhesive having a pH of 5.8 had a gel time ofapproximately 20 minutes. The method described herein for determiningthe gelation time of a bioadhesive is broadly applicable for otherformulations.

Advantageously, bioadhesives of the present disclosure have a gelationtime that is sufficient for allowing the preparation of the formulationand use of the formulation in occluding a vessel. Sufficient times willvary depending on a number of factors such as the type of vesselcontemplated for occlusion and the particular indication for thebioadhesive. In one aspect of the present disclosure, the gelation timeof the bioadhesive is between about 0.5 minute and about 40 minutes,e.g., between about 5 min and about 30 minutes. In other aspects of thepresent disclosure, the gelation time is more than about 5 minutes.Gelation time is measured from the time when mixing is complete, whichtakes about 10 seconds.

The gelation time of the bioadhesives of the present disclosure dependon several factors, including: the composition of the formulation, theconcentration of the components of the formulation, and the pH of theformulation. While several factors influence gelation time, the pH ofthe formulation is intended to be the principal factor that affectsgelation time for application in small blood vessels. For example,bioadhesives that are more acidic tend to have greater gelation timesince the crosslinking reaction between the chemically reactive amine ofthe biopolymer and second chemically reactive groups of crosslinker tendto take longer in acidic media. Hence, the gelation time of thebioadhesive can be adjusted by adjusting the pH of the formulation. Inone aspect of the present disclosure, the pH of the bioadhesives of thepresent disclosure is less than 7.4, e.g., the pH of the formulationsare between about 6.5 and about 5.0 such as between about 6.5 and 5.5.The pH of the formulation can readily be adjusted by adjusting the pH ofany of the components used to prepare the formulation such as adjustingthe pH of a formulation including the biopolymer or adjusting the pH ofa formulation including the crosslinker or by adding a separate bufferwith a particular pH to the formulation.

The concentration of the components of the bioadhesive can be varied. Incertain embodiments of the present disclosure, the concentration of thebiopolymer in the bioadhesive is less than 15% by weight of the totalcontents of the formulation (wt %), e.g., the concentration of thebiopolymer in the bioadhesive is between 7.5 wt % to 5 wt %. Theconcentration of the biocompatible crosslinker can be between 2 wt % to5 wt % of the bioadhesive, e.g. from between 3 wt % to 4 wt %, and theconcentration of the biocompatible rheological modifier can be between0.3 wt % to 1 wt % of the bioadhesive, e.g. from between 0.6 wt % to 0.9wt %.

The bioadhesive can be prepared by combining: (i) a biopolymer havingone or more first chemically reactive amine groups; (ii) a biocompatiblecrosslinker having at least two second chemically reactive groups thatcan chemically react with the one or more first chemically reactiveamine groups of the biopolymer; and (iii) a biocompatible rheologicalmodifier. The biopolymer can be in separate formulations, e.g., inseparate aqueous formulations, which can optionally further includeother additives such as preservatives and/or pH modifiers. Thebiocompatible rheological modifier can be in a separate formulation,e.g., an aqueous formulation, alone or with other additives such aspreservatives and/or pH modifiers or the biocompatible rheologicalmodifier can be included in either the biopolymer formulation or thecrosslinker formulation, or in both.

In an embodiment of the present disclosure, the biopolymer and thebiocompatible rheological modifier can be combined as a singleformulation and the bioadhesive can be prepared by combining thebiopolymer/rheological modifier formulation with a formulation includingthe crosslinker. The crosslinker formulation can also include a buffer,such as citric acid and disodium hydrogen phosphate (also referred to assodium phosphate dibasic or dibasic phosphate), in bacteriostatic water,sterile water, or water for injection (WFI). For example, a bioadhesiveof the present disclosure can be prepared by mixing the biopolymer andthe biocompatible rheological modifier in a first container; mixing thebiocompatible crosslinker in a buffer solution in a second container;and mixing the contents of the first container and the second containertogether to form the bioadhesive.

In certain embodiments, the crosslinker/buffer formulation can beadjusted to have a pH such that the overall pH of the formulation isless than 7.4 when the crosslinker/buffer formulation is combined withthe other components of the bioadhesive. For example, e.g. thecrosslinker/buffer formulation can have a pH of between 7.4 and 4.5,e.g., a pH between 6.5 and 5.0 such as a pH of between 6.5 and 5.5. Byadjusting the pH of the crosslinker/buffer formulation, the gelationtime of the formulation can be adjusted.

When the biopolymer, biocompatible crosslinker and biocompatiblerheological modifier of the present disclosure are combined, the aminegroups of the biopolymer chemically react with the reactive groups ofthe crosslinker over a period of time to form a crosslinked polymericnetwork to form the bioadhesive gel that includes the rheologicalmodifier. FIG. 1 schematically illustrates the structure of forming abioadhesive gel from a bioadhesive of the present disclosure after theformulation had been inserted, e.g., injected, into a blood vessel. Asillustrated in FIG. 1, the biopolymer and crosslinker chemically reactto form biopolymer-crosslinker polymeric network 10. Thebiopolymer-crosslinker polymeric network 10 can also be attached to thesurrounding wall of the vessel through the reactive groups of thecrosslinker. The biocompatible rheological modifier 20 is also includedwith the bioadhesive, and within the bioadhesive gel for a time aftergelation, in the blood vessel 30. The biocompatible rheological modifierhas a viscosity sufficient to facilitate the displacement of blood in ablood vessel and substantially prevents blood flow back into the vesselfor a time sufficient for the biopolymer and crosslinker to form acrosslinked network within the vessel to form a bioadhesive gel whichcan eventually be absorbed into surrounding tissue. However, thebiocompatible rheological modifier also has shear thinning properties toenable the bioadhesive to be injected through a thin gauge needle, suchas a 30 gauge needle.

Biopolymers of the present disclosure include those having one or morefirst chemically reactive amine groups. Preferably the biopolymer hasmultiple chemically reactive amine groups such as multiple primary aminegroups that can chemically react with the crosslinker. Such biopolymersinclude proteins and polysaccharide having one or more first chemicallyreactive amine groups, for example, proteins and polysaccharides havingmultiple primary amine groups. Proteins useful as biopolymers include,for example, albumin and collagen having one or more first chemicallyreactive groups. Polysaccharides useful as biopolymers of the presentdisclosure include chitosans having one or more first chemicallyreactive groups, e.g., chitosans having multiple primary amines as thefirst chemically reactive amine groups. Other biopolymers useful for thepresent disclosure include polyaminoacids having one or more firstchemically reactive amine groups such as polylysine, etc. Thebiopolymers of the present disclosure are not restricted to polymersthat are naturally available but can also include synthetic polymerssuch as amine functionalized polyethylene glycols, polyallylamine andbranched polyethylenimine, for example. In certain embodiments, somecombinations of these or other biopolymers may also be used.

Biocompatible crosslinkers of the present disclosure include thosehaving at least two second chemically reactive groups that canchemically react with the one or more first chemically reactive aminegroups of the biopolymer. Preferably the biocompatible crosslinker hasat least three, four, five, six, seven, eight or more second chemicallyreactive groups that can chemically react with the one or more firstchemically reactive amine groups of the biopolymer. In one aspect of thepresent disclosure, the biocompatible crosslinker is a polyethyleneglycol having chemically reactive N-hydroxysuccinimide (NETS) groupswhich can chemically react with the amines of the biopolymer.

In one aspect of the present disclosure, the biocompatible crosslinkeris a multi-arm polyethylene glycol (PEG) having at least twoN-hydroxysuccinimide (NETS) ester groups as the second chemicallyreactive groups. Such crosslinkers include, for example, 4-arm and 8-armN-hydroxy substituted succinimidyl-PEG (NHS:PEG) crosslinkers.

Biocompatible rheological modifiers of the present disclosure includethose that can increase the viscosity of the bioadhesive. Preferably,the rheological modifier exhibits shear thinning effects; that is, theviscosity of the bioadhesive including the rheological modifier has alower viscosity when a shear force is applied to the formulation. Such aforce can occur when the bioadhesive is ejected from a syringe orsimilar device for inserting the bioadhesive into a vessel.

In one aspect of the present disclosure, the rheological modifierfacilitates the displacement of blood in a blood vessel for a timesufficient for the biopolymer and crosslinker to form a crosslinkednetwork without being washed away or diluted. Preferably, thebiocompatible rheological modifier does not substantially react with thebiopolymer or the biocompatible crosslinker. Rather, the biopolymer andthe crosslinker react with each other within the rheological modifier asa gel within a gel.

Useful rheological modifies of the present disclosure include any one ormore of polysaccharides, alginates, celluloses (including carboxymethyl,hydroxypropylmethyl, and microcrystalline cellulose), gums (such asxanthan gum and guar gum), dextrans, and biocompatible derivatives andbiocompatible salts thereof, as well as some combinations thereof. Thebiocompatible rheological modifiers are distinct from the biopolymers inthat the biocompatible rheological modifiers do not contain anyappreciable number of chemically reactive amines and are chosen suchthat the biocompatible rheological modifier does not substantially reactwith the biopolymer or the biocompatible crosslinker.

Preferably the rheological modifier is a biocompatible polysaccharidesuch as hyaluronic acid or a salt thereof, e.g., sodium hyaluronate.Hyaluronic acid, also known as hyaluronan and HA, is a naturallyoccurring, water soluble polysaccharide, specifically aglycosaminoglycan, which is a major component of the extra-cellularmatrix and is widely distributed in animal tissues. HA and salts thereofhave excellent biocompatibility and can be readily absorbed into thebody when implanted into a mammal. In addition, HA and salts thereof canincrease the viscosity of the bioadhesive and assist in displacing bloodwhen injected into a blood vessel, while having shear thinningproperties when injected through a needle.

In practicing certain embodiments, the rheological modifier should haveor causes a non-sheared viscosity sufficient to displace blood and keepthe blood from pushing back into a treatment area while the biopolymerand crosslinker crosslink. Blood has a viscosity around 3 cp (0.003Pa·s). Glycerin, which is a chemical sometimes used within bloodvessels, has a viscosity around 10 cp (0.01 Pa·s) when in a 72% w/vglycerin/water solution. However, that viscosity allows blood to bleedback into the treatment area quickly, which would not allow enough timefor a biopolymer and crosslinker to crosslink. Thus, a rheologicalmodifier with greater viscosity should be used to enable enough time forthe biopolymer and crosslinker to crosslink, while preferably exhibitingshear thinning properties to enable the bioadhesive to be injectedthrough a thin gauge needle. For example, the biocompatible rheologicalmodifier may be a shear thinning fluid with a non-sheared viscositybetween 500 cp (0.5 Pa·s) and 200,000 cp (200 Pa·s), e.g., between10,000 cp (10 Pa·s) to 200,000 cp (200 Pa·s). One such rheologicalmodifier can include hyaluronic acid or salt thereof as an aqueoussolution. Hyaluronic acid or salt thereof may be prepared in an aqueous1% w/v concentration which will have a non-shear viscosity around100,000 cp (100 Pa·s) depending on the molecular weight of thehyaluronic acid used.

In practicing certain embodiments of the present disclosure, thebioadhesive can be inserted, such as by injection through a needle, intoa vessel, such as a blood vessel, to occlude the vessel. The bioadhesiveof the present disclosure includes: (i) a biopolymer having one or morefirst chemically reactive amine groups; (ii) a biocompatible crosslinkerhaving at least two second chemically reactive groups that canchemically react with the one or more first chemically reactive aminegroups of the biopolymer; and (iii) a biocompatible rheologicalmodifier. As the bioadhesive is injected into the blood vessel, thebioadhesive displaces the blood. After the bioadhesive is insertion intothe vessel, the rheological modifier prevents blood from flowing backinto the treated area while the biopolymer crosslinks with thebiocompatible crosslinker to form a bioadhesive gel, and the crosslinkednetworked can attach to the vessel walls through reactions with thebiopolymer and/or crosslinker, thereby forming an occlusion in thevessel. Once crosslinked, the bioadhesive gel may have a color thatcannot be seen through the skin, and may have a tactile feel similar tothe vessel into which the bioadhesive was injected. Over time, thebiocompatible rheological modifier will be absorbed into the surroundingtissue, as will the bioadhesive gel occlusion and the vessel.

In certain embodiments, the bioadhesive can be prepared by combining (i)a formulation including a biopolymer having one or more first chemicallyreactive amine groups and a biocompatible rheological modifier with (ii)a formulation including a biocompatible crosslinker having at least twosecond chemically reactive groups that can chemically react with the oneor more first chemically reactive amine groups. The formulationincluding the biocompatible crosslinker can further include a bufferhaving a pH of between 7.4 and 4.5, e.g., a pH of between 6.5 and 5.2.

As explained above, adjusting the pH of the bioadhesive such as byadjusting the pH of one or more of the component formulations to preparethe bioadhesive, can adjust the gelation time of the formulation. Thegelation time of the formulation can be adjusted such that it willcrosslink quickly, for example, after about half a minute, or moreslowly, such as after about 5 minutes. The gelation time can be adjustedso that the bioadhesive can range from about 1 minute to about 40minutes, more preferably between about 5 minutes and 40 minutes. Whenthe gelation time is greater than 5 minutes, the bioadhesive isparticularly suited for the treatment of superficial or spider bloodvessels. The formulations can also be broadly used to treat larger bloodvessels (perforators, varicose veins, arteries), vascular malformations(aneurysms, AVMs, fistula sealant, etc.), and, for the faster gelationtimes, for wound closure applications.

FIGS. 2 and 3 illustrate properties of a bioadhesive of the presentdisclosure useful for occluding blood vessels. As shown in FIG. 2, thebioadhesive can be injected into a blood vessel for a period of about 20to 25 minutes after the components of the bioadhesive are combined andtransferred into a syringe. During this injection time period, the forceneeded to eject the formulation from the syringe is similar to the forceneeded to eject a 50% (w/v) and 72% (w/v) glycerin/water solution, i.e.,between about 7 N and 17 N, through the same size needle. The 50% (w/v)and 72% (w/v) glycerin/water solutions represent the properties of otherfluids currently used to treat blood vessels by injection through aneedle into the blood vessel. After about 25 minutes, the formulationbecomes increasingly more difficult to eject from the syringe due to theformation of the crosslinked polymer.

FIG. 2 further illustrates that during the first 20 minute time periodof this bioadhesive embodiment, the formulation can be prepared and theninjected into a blood vessel through a needle, such as a 30 gaugeneedle. During the first 20 minutes, the biocompatible rheologicalmodifier will displace blood, hold the bioadhesive in place, and preventblood from washing the formulation away. After 20 to 30 minutes, theinjected formulation crosslinks in the blood vessel and is more or lessfully formed as an occlusion such that the patient can be released fromthe treatment.

FIG. 3 shows how adjusting the pH of a formulation including thecrosslinker prior to combining the crosslinker formulation with abiopolymer and rheological modifier can change the gelation time. Asshown in FIG. 3, the gelation time can be adjusted from about 40 minutesto about 5 minutes. Short gelation times are advantageous for applyingthe bioadhesive of the present disclosure to, for example large bloodvessels or fistula sealing.

In an aspect of the present disclosure, a bioadhesive is injected into ablood vessel of a mammal, such as a human, to occlude the blood vessel.The methods of the present disclosure are applicable to injecting theformulation into a blood vessel having a diameter of between 0.3 mm to 6mm and injecting the formulation into a blood vessel having a diameterof greater than 6 mm. The injected bioadhesive can rapidly andcompletely occlude the vessel followed by absorption of the treatedvessel and the bioadhesive gel occlusion into surrounding tissue overtime.

In another aspect of the present disclosure, the components of thebioadhesive can be provided in a kit. For example, the kit can comprise:(i) a first container including a biopolymer having one or more firstchemically reactive amine groups; (ii) a second container including abiocompatible crosslinker having at least two second chemically reactivegroups that can chemically react with the one or more first chemicallyreactive amine groups of the biopolymer; and (iii) a biocompatiblerheological modifier. The biocompatible rheological modifier canadvantageously be included in either the first container or the secondcontainer. Alternatively, the biocompatible rheological modifier can beincluded in a third container such that the kit would comprise threeseparate containers. The kit can also include another containerincluding a buffer solution. The buffer solution can be used foradjusting the pH of the components of the bioadhesive prior to preparingthe formulation or can be added directly to the formulation preparedfrom the biopolymer, crosslinker and rheological modifier.

The kit can also include instructions on how to combine the variouscomponents of the kit to prepare and apply the bioadhesive. Theinstructions can be included as an insert, incorporated into thecontainer(s) and/or in the packaging of the kit.

In an embodiment of the present disclosure, the kit includes: (i) afirst container, e.g., a syringe, including albumin having one or morefirst chemically reactive amine groups; (ii) a second containerincluding a multi-arm polyethylene glycol having at least two second,e.g., at least three, chemically reactive groups that can chemicallyreact with the one or more first chemically reactive amine groups of thealbumin; and (iii) a hyaluronate salt rheological modifier includedeither in the first container or the second container or in a thirdcontainer.

Examples

The following examples are intended to further illustrate certainpreferred embodiments of the invention and are not limiting in nature.Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific substances and procedures described herein.

Biomaterials: Recombinant Human Albumin was obtained from Invitria (FortCollins, Colo.) as a 25% stock solution (Albipur DF) or as a powder(Albumin DX LR). Polyethylene Glycol (PEG) crosslinker with a molecularweight of 15 kDa was obtained from Jenkem (Plano, Tex.). Two forms of an8-arm N-hydroxy substituted succinimidyl-PEG (NHS:PEG) crosslinker wereused, which were an 8-arm PEG Succinimidyl Succinate (8ARM-PEG-SS) or an8-arm PEG Succinimidyl Glutarate (8ARM-PEG-SG). Sodium Hyaluronate(Pharm Grade 150 or Pharm Grade 80) was obtained from FMC Corporation(Novamatrix) (Sandvika, Norway).

Buffers and Preservatives: Citric acid and dibasic sodium phosphatebuffer salts were obtained from Sigma Aldrich (St Louis, Mo.). Sterile1× phosphate buffered saline (PBS) was obtained from Fisher Scientific(Waltham, Mass.). Sodium octanoate and N-acetyl tryptophan preservativecompounds were obtained from Sigma Aldrich. Sodium octanoate andN-acetyl tryptophan can be added to a formulation for sterilization andshelf-life extension.

Preparation of Polymer Blends

A sodium hyaluronate solution was prepared by combining 1.5 g of sodiumhyaluronate with 100 mL of 1X PBS in a beaker with constant stirring at600 RPM at 4° C. for an approximate 12 hour period. An albumin solutionwas prepared by combining 25 g of albumin in a beaker with 100 mL ofwater for injection (WFI) grade water with constant stirring at 600 RPMat 4° C. over a period of about 12 hours. Sodium hyaluronate/albuminformulations were prepared, for example, by either: (a) adding 60 mL ofAlbumin solution (25% w/v) to 100 mL of sodium hyaluronate solution(1.5% w/v) with stirring at 300 RPM for 4 h, or (b) adding 33 mL ofAlbumin solution (25% w/v) to 100 mL of sodium hyaluronate solution(1.5% w/v) with stirring at 300 RPM for 4 h.

Preparation of Citrate-Phosphate Buffers for 8-Arm 15 kDa NHS:PEG

A 0.1M citric acid solution stock in WFI grade water was prepared and a0.2M Dibasic sodium phosphate solution stock in WFI grade water was alsoprepared. Buffer solutions were prepared from these stock solutions bymixing the citric acid and dibasic sodium phosphate stock solutions atcertain volume ratios to obtain a target pH for the buffer. The pH ofthe buffer governs the gelation time of the bioadhesive. Table 1 ispresented below for the volume ratio and target pH for a bioadhesiveincluding pH of 5.2, 5.8 and 6.5.

TABLE 1 0.2M Dibasic sodium 0.1M citric phosphate (ml) acid (ml) pH 5.444.6 2.6 7.8 42.2 2.8 10.2 39.8 3.0 12.3 37.7 3.2 14.1 35.9 3.4 16.133.9 3.6 17.7 32.3 3.8 19.3 30.7 4.0 20.6 29.4 4.2 22.2 27.8 4.4 23.326.7 4.6 24.8 25.2 4.8 25.7 24.3 5.0 26.7 23.3 5.2 27.8 22.2 5.4 29.021.0 5.6 30.3 19.7 5.8 32.1 17.9 6.0 33.1 16.9 6.2 34.6 15.4 6.4 36.413.6 6.6 40.9 9.1 6.8 43.6 6.5 7.0

A crosslinker buffered solution was prepared by combining 0.15 g of8-arm NHS:PEG and 1 mL of any of a citric acid and dibasic sodiumphosphate buffer solution. The volume ratio of the citric acid anddibasic sodium phosphate will determine the pH of the solution asprovided in the table above. It is preferable that the crosslinkerbuffered solution is prepared immediately prior to use in preparing abioadhesive gel.

Bioadhesives Including HA

Bioadhesives were prepared by mixing a biopolymer, a biocompatiblecrosslinker and a biocompatible rheological modifier, as describedabove. In this example, 4 mL of the polymer blend described above wastransferred into a 5 mL syringe. A solution of 1 mL of the NHS:PEGdissolved in a citrate-phosphate buffer was transferred into another 5mL syringe. The two syringes were then connected to each other by a lueradapter and the contents of the syringes were mixed by moving thecontents of the two syringes back and forth for about 10 times toprepare the bioadhesive. The entire 5 mL formulation was thentransferred to one of the syringes and the second syringe removedleaving the luer adapter attached to the syringe with the 5 mL offormulation. The bioadhesive was then transferred to 5 syringes of 1 mLeach for injections through a 30 gauge needle.

Bioadhesive Gel

Several bioadhesive gels were prepared by combining a first formulationincluding albumin and sodium hyaluronate with a second formulationincluding an 8-arm NHS:PEG with citric acid and dibasic sodium phosphatebuffers. The various bioadhesive gels were prepared from variouscrosslinker formulations having a buffer pH range of between 6.5 and5.2, which was measured as described below.

Control of the Rate of Gelation

The rate of formation of the cross-linked network of albumin and NHS:PEG(i.e., gelation) can be controlled by the pH of the bioadhesive. Toincrease the rate of cross-linking, the pH of the formulation isincreased, and conversely, to decrease the rate of cross-linking, the pHof the formulation is decreased. The pH of the formulation is controlledprincipally by the addition of a buffer in terms of both the bufferstrength and buffer pH.

FIG. 3 shows the effect of pH of the bioadhesive on the rate of gelationof a formulation containing 7.5 wt % human serum albumin, 0.75 wt %sodium hyaluronate and 3 wt % 8ARM-PEG in water for injection at roomtemperature. The rate of gelation can also be controlled by adjustingthe concentration of albumin and the cross-linker. The buffer wasincluded in the solution with the 8ARM-PEG. The gelation time is theamount of time required for the formulation to transform from the liquidstate to the cross-linked gel state (as defined above). An advantage ofthe present disclosure is that the onset of any appreciable crosslinkingcan be delayed. As shown in FIG. 3, the gelation time can be adjustedfrom approximately 20 minutes for a bioadhesive having a pH of 5.8 toapproximately 10 minutes for a bioadhesive having a pH of 6.0 bychanging the pH of the bioadhesive.

As also shown in Tables 2 and 3 below, the gelation time can be changedby changing either the pH of the bioadhesive formulation or theconcentration of the biopolymer. The data in Tables 2 and 3 below areplotted in FIG. 4, which graphically illustrates the effect of pH andconcentration of biopolymer on approximate gelation times. Thebioadhesives for Tables 2 and 3 were prepared and measured as follows.

Stock solutions of 25% w/v Recombinant Human Albumin (pH 6.5) and 1.5%w/v Sodium Hyaluronate (pH 6.5) were used to prepare formulations asshown in Table 2 below.

TABLE 2 Albumin Sodium Hyaluronate pH pH Gel time (% w/v) (% w/v) bufferformulation (min) 5 0.9 5.2 5.8 34 5 0.9 5.8 6 20 5 0.9 6.5 6.3 12 6.50.81 5.2 5.8 22 6.5 0.81 5.8 6 16 6.5 0.81 6.5 6.3 6 7.5 0.75 5.2 5.8 207.5 0.75 5.8 6 10 7.5 0.75 6.5 6.3 5 7.5 0.75 7 6.8 2 7.5 0.75 7.4 7 1.5

Stock solutions of 25% w/v Bovine Serum Albumin (pH 7.0) and 1.5% w/vSodium Hyaluronate (pH 7.0) were used to prepare formulations as shownin Table 3 below.

TABLE 3 Albumin Sodium Hyaluronate pH pH Gel time (% w/v) (% w/v) bufferformulation (min) 10 0.6 4.6 5.5 14 10 0.6 5.2 6 8 10 0.6 5.8 6.3 5.5 100.6 6.5 6.8 2 10 0.6 7.4 7.3 0.5 7.5 0.75 6.5 6.8 2.5 7.5 0.75 4.6 5.525 7.5 0.75 5.2 6 15 6.5 0.81 5.2 6 17 6.5 0.81 5.8 6.3 6 5 0.9 5.8 6.313

The pH of the bioadhesives recorded in the tables above was measuredwith the use of pH test strips having a resolution of 0.5 units. A pHrange was identified based on the color of the strips after it wasimmersed/contacted with the bioadhesive. A mean value (mid-range value)was used to report the pH values in the tables above. Gelation timeswere approximated by observing the bioadhesive in a syringe every 0.5min for any distinct change in rheology and/or opacity of theformulation in the syringe and the bioadhesive was considered gelledwhen a distinct change in rheology and/or opacity of the formulationoccurred. It was further observed that this visual method was equivalentto the quantitative method described above of determining gelation timeby plotting the force over time of the bioadhesive extruded from a 1 mLsyringe through a 30G needle at a fixed flow rate of 1.5 mL/min anddetermining the first inflection point of a sigmoidal curve (i.e., wherethe curve becomes concave up; see FIG. 3) for the formulations tested.

Occluding Blood Vessels

Bioadhesives including HA were injected into a marginal vein of a rabbitto demonstrate rapid and complete vein occlusion followed by controlledabsorption of the treated vein into surrounding tissue over a 30-60 dayperiod. Histological findings confirm the presence of collagenousmaterial in the area where the vein originally was with minimalinflammation. Briefly, formulations were injected into the dorsalmarginal ear vein of rabbits where the size of veins was in the range of0.3-2 mm diameter. Injections were performed using a 30G needle and a 1mL syringe. Blood pressure in the veins was reduced (to mimic spidervein physiological conditions) by either applying compression distallyor by partially occluding the medial artery that supplies blood to theear. Upon injection, the formulation displaced blood in the vein andgelled over the period of time based on the pH of the formulation andthe % w/v of either the biopolymer component or the cross-linker. It maybe noted that gel time within a vessel (in-situ) is likely to be fasterthan measurements reported above as physiological pH is 7.4 and theformulation is in contact with physiological fluid at that pH uponinjection.

Bioadhesives Including HPMC

Albumin was sourced and prepared as described above in a 25% stocksolution. An 8-ARM NHS:PEG was reconstituted using a citrate-phosphatebuffer. A 4% w/v stock solution of hydroxypropylmethyl cellulose (HPMC)from Sigma Aldrich (St. Louis, Mo.) was prepared in phosphate bufferedsaline (PBS) as a rheological modifier. A 1.5% w/v sodium hyaluronatesolution was also prepared as described above. The followingbioadhesives were prepared with 3% w/v NHS:PEG, and evaluated as havingthe specified pH and gelation times:

Albumin HPMC HA pH Gel Time (% w/v) (% w/v) (% w/v) formulation (min)7.5 2 0 6.5 5 7.5 1 0 6.0 10 7.5 0.4 0.45 6.75 3

Each of these bioadhesives was injectable through a 30G needle using a 1mL syringe.

Bioadhesives Including Chitosan

Chitosan Chloride (Protasan UP CL 113) was obtained from Novamatrix (asubsidiary of FMC) (Sandvika, Norway) and prepared as a 4% w/v solutionin phosphate buffered saline. An 8-ARM NHS:PEG was reconstituted using aphosphate buffered saline. A 4% w/v stock solution ofhydroxypropylmethyl cellulose (HPMC) from Sigma Aldrich (St. Louis, Mo.)was prepared in phosphate buffered saline as a rheological modifier. Thefollowing bioadhesives were prepared with 3% w/v NHS:PEG, and evaluatedas having the specified pH and gelation times:

Chitosan HPMC pH Gel Time (% w/v) (% w/v) formulation (min) 1.2 1.0 6.045 1.6 0.8 6.5 40

Each of these bioadhesives was injectable through a 30G needle using a 1mL syringe.

Bioadhesives Including Collagen

Porcine Type I collagen was obtained from Sofradim Production (Trevoux,France), and prepared in 0.1M citric acid and adjusted to a pH of 6.5with 0.2M sodium hydroxide. A 1.5% w/v sodium hyaluronate solution wasalso prepared as described above. A 4% w/v HPMC solution was prepared asdescribed above. An 8-ARM NHS:PEG was reconstituted using a phosphatebuffered saline. The following bioadhesives were prepared with 3% w/vNHS:PEG, and evaluated as having the specified pH and gelation times:

Collagen Rheological Modifier pH Gel Time (% w/v) (% w/v) formulation(min) 1.8 HA - 0.3 6.5 7 1.8 HPMC - 0.4 6.5 7

Each of these bioadhesives was injectable through a 30G needle using a 1mL syringe.

Only the preferred embodiments of the present invention and examples ofits versatility are shown and described in the present disclosure. It isto be understood that the present invention is capable of use in variousother combinations and environments and is capable of changes ormodifications within the scope of the inventive concept as expressedherein. Thus, for example, those skilled in the art will recognize, orbe able to ascertain, using no more than routine experimentation,numerous equivalents to the specific substances, procedures andarrangements described herein. Such equivalents are considered to bewithin the scope of this invention, and are covered by the followingclaims.

1: A method of treating a vascular malformation of a mammal, the methodcomprising: injecting a bioadhesive into a vascular malformation of amammal to displace blood in the malformation and prevent blood flow backinto the malformation and to crosslink the bioadhesive in themalformation to form an occlusion in the malformation, wherein thebioadhesive comprises: (i) a biopolymer having one or more firstchemically reactive amine groups; (ii) a biocompatible crosslinkerhaving at least two second chemically reactive groups that canchemically react with the one or more first chemically reactive aminegroups of the biopolymer; and (iii) a biocompatible rheologicalmodifier, and wherein the biopolymer and crosslinker form a crosslinkednetwork and the biocompatible rheological modifier does notsubstantially react with the biopolymer or the biocompatiblecrosslinker. 2: The method of claim 1 wherein the biocompatiblerheological modifier has a viscosity sufficient to displace fluid withinthe malformation while the biopolymer and biocompatible crosslinkercrosslink. 3: The method of claim 1 comprising injecting the bioadhesiveinto the malformation by a syringe. 4: The method of claim 1 furthercomprising preparing the bioadhesive by combining the biopolymer, thebiocompatible crosslinker and the biocompatible rheological modifierprior to injecting the bioadhesive into the malformation.
 5. The methodof claim 1 wherein the bioadhesive has a gelation time of between 0.5minute and 40 minutes. 6: The method of claim 1, wherein the rheologicalmodifier displaces blood and prevents blood flow back into the bloodmalformation while the biopolymer crosslinks with the biocompatiblecrosslinker. 7: The method of claim 1, wherein the rheological modifieris absorbed into tissue surrounding the blood malformation after thebioadhesive has crosslinked. 8: The method of claim 1 wherein thebiocompatible rheological modifier is a shear thinning fluid with anon-sheared viscosity between 0.5 Pa·s and 200 Pa·s. 9: The method ofclaim 1 wherein the biopolymer is an albumin having one or more primaryamines as the first chemically reactive amine groups, the biocompatiblecrosslinker is a multi-arm PEG having at least two or moreN-hydroxysuccinimide ester groups as the second chemically reactivegroups and the biocompatible rheological modifier is sodium hyaluronate.10: The method of claim 1 wherein the biopolymer is a protein or apolysaccharide having one or more primary amines as the first chemicallyreactive groups. 11: The method of claim 1 wherein the biopolymer isalbumin. 12: The method of claim 1 wherein the biocompatible crosslinker is a multi-arm polyethylene glycol (PEG) having at least two ormore N-hydroxysuccinimide (NHS) ester groups as the second chemicallyreactive groups. 13: The method of claim 1 wherein the biocompatiblerheological modifier is hyaluronic acid or a salt thereof. 14: Themethod of claim 13 wherein the hyaluronic acid or salt thereof has anon-sheared viscosity between 0.5 Pa·s and 200 Pa·s. 15: The method ofclaim 1 wherein the concentration of the biopolymer in the bioadhesiveis less than 15 wt %. 16: The method of claim 1 wherein the bioadhesivehas a pH of less than 7.4. 17: The method of claim 1 wherein thebiopolymer crosslinks with the biocompatible crosslinker to form abioadhesive gel. 18: The method of claim 1, wherein the bioadhesive hasa gelation time of between 5 minutes and 40 minutes. 19: The method ofclaim 1 wherein the bioadhesive has a pH of between about 6.5 and about5.0. 20: The method of claim 1 wherein the occluded malformation isabsorbed into surrounding tissue over time after injection of thebioadhesive into the malformation. 21: The method of claim 1 wherein thevascular malformation comprises an aneurysm. 22: The method of claim 1wherein the vascular malformation comprises an AVM. 23: The method ofclaim 1 wherein the vascular malformation comprises a fistula. 24: Themethod of claim 1 wherein the biopolymer comprises chitosan. 25: Themethod of claim 1 wherein the biocompatible crosslinker comprisesgenipin. 26: The method of claim 1 wherein the rheological modifiercomprises hydroxypropylmethyl cellulose (HPMC).